Protein kinase C inhibits Kv1.1 potassium channel function

The regulation by protein kinase C (PKC) of recombinantvoltage-gated potassium (K) channels in frog oocytes was studied. Phorbol 12-myristate 13-acetate (PMA; 500 nM), an activator of PKC,caused persistent and large (up to 90%) inhibition of mouse, rat, andfly Shaker K currents. K currentinhibition by PMA was blocked by inhibitors of PKC, and inhibition wasnot observed in control experiments with PMA analogs that do notactivate PKC. However, site-directed substitution of potential PKCphosphorylation sites in the Kv1.1 protein did not prevent currentinhibition by PMA. Kv1.1 current inhibition was also not accompanied bychanges in macroscopic activation kinetics or in theconductance-voltage relationship. In Western blots, Kv1.1 membraneprotein was not significantly reduced by PKC activation. The injectionof oocytes with botulinum toxin C3 exoenzyme blocked the PMA inhibitionof Kv1.1 currents. These data are consistent with the hypothesis thatPKC-mediated inhibition of Kv1.1 channel function occurs by a novelmechanism that requires a C3 exoenzyme substrate but does not alterchannel activation gating or promote internalization of the channel protein.

     

Actinomycin D-induced apoptosis involves the potassium channel Kv1.3

Several cytostatic agents are known to induce apoptosis in T-leukemic cells. Although a variety of studies show the central role of apoptosis in cytostatic drug-induced cell death, many molecular details require definition. Here, we demonstrate that cells genetically deficient for the potassium channel Kv1.3 are resistant to apoptosis initiated by the cytostatic drug actinomycin D. Retransfection of Kv1.3 restores sensitivity of the cells to actinomycin D. Cells lacking Kv1.3 fail to respond to actinomycin D with DNA fragmentation, release of cytochrome c, and loss of mitochondrial membrane potential (Delta Psi(m)), while cells functionally expressing Kv1.3 rapidly undergo those changes indicative for apoptosis. The data indicate a central role of the ion channel Kv1.3 in actinomycin D-triggered apoptosis.     

State-dependent inactivation of the Kv3 potassium channel.

Inactivation of Kv3 (Kv1.3) delayed rectifier potassium channels was studied in the Xenopus oocyte expression system. These channels inactivate slowly during a long depolarizing pulse. In addition, inactivation accumulates in response to a series of short depolarizing pulses (cumulative inactivation), although no significant inactivation occurs within each short pulse. The extent of cumulative inactivation does not depend on the voltage during the depolarizing pulse, but it does vary in a biphasic manner as a function of the interpulse duration. Furthermore, the rate of cumulative inactivation is influenced by changing the rate of deactivation. These data are consistent with a model in which Kv3 channel inactivation is a state-dependent and voltage-independent process. Macroscopic and single channel experiments indicate that inactivation can occur from a closed (silent) state before channel opening. That is, channels need not open to inactivate. The transition that leads to the inactivated state from the silent state is, in fact, severalfold faster then the observed inactivation of current during long depolarizing pulses. Long pulse-induced inactivation appears to be slow, because its rate is limited by the probability that channels are in the open state, rather than in the silent state from which they can inactivate. External potassium and external calcium ions alter the rates of cumulative and long pulse-induced inactivation, suggesting that antagonistic potassium and calcium binding steps are involved in the normal gating of the channel.     

Discovery of triarylethanolamine inhibitors of the Kv1.5 potassium channel

A series of triarylethanolamine inhibitors of the Kv1.5 potassium channel have been prepared and evaluated for their effects in vitro and in vivo. The structure–activity relationship (SAR) studies described herein led to the development of potent, selective and orally active inhibitors of Kv1.5.     

Reduced Kv1.3 potassium channel expression in human prostate cancer

The presence of Kv1.3 voltage-gated potassium channels in rat and human prostate epithelial cells has been previously reported. We examined, by immunohistochemistry, Kv1.3 levels in 10 normal human prostate, 18 benign prostatic hyperplasia (BPH) and 147 primary human prostate cancer (Pca) specimens. We found high epithelial expression of Kv1.3 in all normal prostate, 16 BPH and 77 (52%) Pca specimens. Compared to normal, Kv1.3 levels were reduced in 1 (6%) BPH specimen and in 70 (48%) Pca specimens. We found a significant inverse correlation between Kv1.3 levels and tumor grade (r = -0.25, P = 0.003) as well as tumor stage (r = -0.27, P = 0.001). Study of an additional 30 primary Pca specimens showed that 15 (50%) had reduced Kv1.3 immunostaining compared to matched normal prostate tissue. Our data suggest that in Pca reduced Kv1.3 expression occurs frequently and may be associated with a poor outcome.     

Association of Kv1.5 and Kv1.3 contributes to the major voltage-dependent K+ channel in macrophages

Voltage-dependent K(+) (Kv) currents in macrophages are mainly mediated by Kv1.3, but biophysical properties indicate that the channel composition could be different from that of T-lymphocytes. K(+) currents in mouse bone marrow-derived and Raw-264.7 macrophages are sensitive to Kv1.3 blockers, but unlike T-cells, macrophages express Kv1.5. Because Shaker subunits (Kv1) may form heterotetrameric complexes, we investigated whether Kv1.5 has a function in Kv currents in macrophages. Kv1.3 and Kv1.5 co-localize at the membrane, and half-activation voltages and pharmacology indicate that K(+) currents may be accounted for by various Kv complexes in macrophages. Co-expression of Kv1.3 and Kv1.5 in human embryonic kidney 293 cells showed that the presence of Kv1.5 leads to a positive shift in K(+) current half-activation voltages and that, like Kv1.3, Kv1.3/Kv1.5 heteromers are sensitive to r-margatoxin. In addition, both proteins co-immunoprecipitate and co-localize. Fluorescence resonance energy transfer studies further demonstrated that Kv1.5 and Kv1.3 form heterotetramers. Electrophysiological and pharmacological studies of different ratios of Kv1.3 and Kv1.5 co-expressed in Xenopus oocytes suggest that various hybrids might be responsible for K(+) currents in macrophages. Tumor necrosis factor-alpha-induced activation of macrophages increased Kv1.3 with no changes in Kv.1.5, which is consistent with a hyperpolarized shift in half-activation voltage and a lower IC(50) for margatoxin. Taken together, our results demonstrate that Kv1.5 co-associates with Kv1.3, generating functional heterotetramers in macrophages. Changes in the oligomeric composition of functional Kv channels would give rise to different biophysical and pharmacological properties, which could determine specific cellular responses.     

Structural determinants of Kvbeta1.3-induced channel inactivation: a hairpin modulated by PIP2

Inactivation of voltage-gated Kv1 channels can be altered by Kvbeta subunits, which block the ion-conducting pore to induce a rapid ('N-type') inactivation. Here, we investigate the mechanisms and structural basis of Kvbeta1.3 interaction with the pore domain of Kv1.5 channels. Inactivation induced by Kvbeta1.3 was antagonized by intracellular PIP(2). Mutations of R5 or T6 in Kvbeta1.3 enhanced Kv1.5 inactivation and markedly reduced the effects of PIP(2). R5C or T6C Kvbeta1.3 also exhibited diminished binding of PIP(2) compared with wild-type channels in an in vitro lipid-binding assay. Further, scanning mutagenesis of the N terminus of Kvbeta1.3 revealed that mutations of L2 and A3 eliminated N-type inactivation. Double-mutant cycle analysis indicates that R5 interacts with A501 and T480 of Kv1.5, residues located deep within the pore of the channel. These interactions indicate that Kvbeta1.3, in contrast to Kvbeta1.1, assumes a hairpin structure to inactivate Kv1 channels. Taken together, our findings indicate that inactivation of Kv1.5 is mediated by an equilibrium binding of the N terminus of Kvbeta1.3 between phosphoinositides (PIPs) and the inner pore region of the channel.     

Coupling of voltage-dependent potassium channel inactivation and oxidoreductase active site of Kvbeta subunits

The accessory beta subunits of voltage-dependent potassium (Kv) channels form tetramers arranged with 4-fold rotational symmetry like the membrane-integral and pore-forming alpha subunits (Gulbis, J. M., Mann, S., and MacKinnon, R. (1999) Cell. 90, 943-952). The crystal structure of the Kvbeta2 subunit shows that Kvbeta subunits are oxidoreductase enzymes containing an active site composed of conserved catalytic residues, a nicotinamide (NADPH)-cofactor, and a substrate binding site. Also, Kvbeta subunits with an N-terminal inactivating domain like Kvbeta1.1 (Rettig, J., Heinemann, S. H., Wunder, F., Lorra, C., Parcej, D. N., Dolly, O., and Pongs, O. (1994) Nature 369, 289-294) and Kvbeta3.1 (Heinemann, S. H., Rettig, J., Graack, H. R., and Pongs, O. (1996) J. Physiol. (Lond.) 493, 625-633) confer rapid N-type inactivation to otherwise non-inactivating channels. Here we show by a combination of structural modeling and electrophysiological characterization of structure-based mutations that changes in Kvbeta oxidoreductase activity may markedly influence the gating mode of Kv channels. Amino acid substitutions of the putative catalytic residues in the Kvbeta1.1 oxidoreductase active site attenuate the inactivating activity of Kvbeta1.1 in Xenopus oocytes. Conversely, mutating the substrate binding domain and/or the cofactor binding domain rescues the failure of Kvbeta3.1 to confer rapid inactivation to Kv1.5 channels in Xenopus oocytes. We propose that Kvbeta oxidoreductase activity couples Kv channel inactivation to cellular redox regulation.     

Ceramide inhibits the potassium channel Kv1.3 by the formation of membrane platforms

Previous studies suggested a central role of sphingomyelin- and cholesterol-enriched membrane rafts in the initiation of signaling via many receptors. Here, we investigated the role of membrane rafts for the function of the voltage-gated potassium channel Kv1.3. We demonstrate that Kv1.3 localizes in the cell membrane to pre-existing small, sphingolipid- and cholesterol-enriched membrane rafts. Transformation of these small rafts to large ceramide-enriched membrane platforms was achieved by stimulation of the endogenous acid sphingomyelinase, addition of exogenous sphingomyelinase or treatment of the cells with C(16)-ceramide and resulted in clustering of Kv1.3 within ceramide-enriched membrane platforms and inhibition of the channel's activity. Likewise, disruption of pre-existing small rafts inhibited Kv1.3 activity. This indicates that intact small membrane rafts are required for Kv1.3 activity and an alteration of the lipid environment of rafts inhibits Kv1.3. These data, thus, may suggest a novel concept for the regulation of ion channels by the cell membrane composition.     

Two adaptor proteins differentially modulate the phosphorylation and biophysics of Kv1.3 ion channel by SRC kinase.

The Shaker family K(+) channel protein, Kv1.3, is tyrosine phosphorylated by v-Src kinase at Tyr(137) and Tyr(449) to modulate current magnitude and kinetic properties. Despite two proline rich sequences and these phosphotyrosines contained in the carboxyl and amino terminals of the channel, v-Src kinase fails to co-immunoprecipitate with Kv1.3 as expressed in HEK 293 cells, indicating a lack of direct Src homology 3- or Src homology 2-mediated protein-protein interaction between the channel and the kinase. We show that the adaptor proteins, n-Shc and Grb10, are expressed in the olfactory bulb, a region of the brain where Kv1.3 is highly expressed. In HEK 293 cells, co-expression of Kv1.3 plus v-Src with Grb10 causes a decrease in v-Src-induced Kv1.3 tyrosine phosphorylation and a reversal of v-Src-induced Kv1.3 current suppression, increase in inactivation time constant (tau(inact)), and disruption of cumulative inactivation properties. Co-expression of Kv1.3 plus v-Src with n-Shc did not significantly alter v-Src-induced Kv1.3 current suppression but reversed v-Src induced increased tau(inact) and restored the right-shifted voltage at half-activation (V(1/2)) induced by v-Src. The v-Src-induced shift in V(1/2) and increased tau(inact) was retained when Tyr(220), Tyr(221), and Tyr(304) in the CH domain of n-Shc were mutated to Phe (triple Shc mutant) but was reversed back to control values when either wild-type Shc or the family member Sck, which is not a substrate for Src kinase, was substituted for the triple Shc mutant. Thus the portion of the CH domain that includes Tyr(220), Tyr(221), and Tyr(304) may regulate a shift in Kv1.3 voltage dependence and inactivation kinetics produced by n-Shc in the presence of v-Src. Collectively these data indicate that Grb10 and n-Shc adaptor molecules differentially modulate the degree of Kv1.3 tyrosine phosphorylation, the channel's biophysical properties, and the physical complexes associated with Kv1.3 in the presence of Src kinase.     

Effect of external pH on activation of the Kv1.5 potassium channel

We studied the mechanism by which external acidification from pH 7.3 to 6.8 reduced current magnitude in the Kv1.5 potassium channel. At physiological external [K(+)], a shift in the voltage-dependence of activation was entirely responsible for the acidification-induced decrease in Kv1.5 current magnitude (pK = 7.15). Elevation of external [Ca(2+)] or [Mg(2+)] identically shifted activation curves to the right and identically shifted the pH-sensitivity of the activation curves to more acidic values. Similar observations were made with the Kv2.1 K(+) channel, except that the pK for the activation shift was out of the physiological range. These data are consistent with a mechanism by which acidification shifted activation via modification of a local surface potential. Elimination of eight positive charges within the outer vestibule of the conduction pathway had no effect on the voltage-dependence of activation at pH 7.3 or higher, which suggested that sites exposed to the conduction pathway within the outer vestibule did not directly contribute to the relevant local surface potential. However, mutations at position 487 (within the conduction pathway) displaced the pK of the pH-sensitive shift in activation, such that the sensitivity of Kv1.5 current to physiologically relevant changes in pH was reduced or eliminated. These results suggest that, among voltage-gated K(+) channels, activation in Kv1.5 is uniquely sensitive to physiologically relevant changes in pH because the pK for the sites that contribute to the local surface potential effect is near pH 7. Moreover, the pK for the activation shift depends not only on the nature of the sites involved but also on structural orientation conferred, in part, by at least one residue within the conduction pathway.     

Lactam sulfonamides as potent inhibitors of the Kv1.5 potassium ion channel

A series of lactam sulfonamides has been discovered and optimized as inhibitors of the Kv1.5 potassium ion channel for treatment of atrial fibrillation. In vitro structure–activity relationships from lead structure C to optimized structure 3y are described. Compound 3y was evaluated in a rabbit PD-model and was found to selectively prolong the atrial effective refractory period at submicromolar concentrations.     

Modulation of Kv1.5 potassium channel gating by extracellular zinc.

Zinc ions are known to induce a variable depolarizing shift of the ionic current half-activation potential and substantially slow the activation kinetics of most K(+) channels. In Kv1.5, Zn(2+) also reduces ionic current, and this is relieved by increasing the external K(+) or Cs(+) concentration. Here we have investigated the actions of Zn(2+) on the gating currents of Kv1.5 channels expressed in HEK cells. Zn(2+) shifted the midpoint of the charge-voltage (Q-V) curve substantially more (approximately 2 times) than it shifted the V(1/2) of the g-V curve, and this amounted to +60 mV at 1 mM Zn(2+). Both Q1 and Q2 activation charge components were similarly affected by Zn(2+), which indicated free access of Zn(2+) to channel closed states. The maximal charge movement was also reduced by 1 mM Zn(2+) by approximately 15%, from 1.6 +/- 0.5 to 1.4 +/- 0.47 pC (n = 4). Addition of external K(+) or Cs(+), which relieved the Zn(2+)-induced ionic current reduction, decreased the extent of the Zn(2+)-induced Q-V shift. In 135 mM extracellular Cs(+), 200 microM Zn(2+) reduced ionic current by only 8 +/- 1%, compared with 71% reduction in 0 mM extracellular Cs(+), and caused a comparable shift in both the g-V and Q-V relations (17.9 +/- 0.6 mV vs. 20.8 +/- 2.1 mV, n = 6). Our results confirm the presence of two independent binding sites involved in the Zn(2+) actions. Whereas binding to one site accounts for reduction of current and binding to the other site accounts for the gating shift in ionic current recordings, both sites contribute to the Zn(2+)-induced Q-V shift.     

Effects of changes in extracellular pH and potassium concentration on Kv1.3 inactivation

The Kv1.3 channel inactivates via the P/C-type mechanism, which is influenced by a histidine residue in the pore region (H399, equivalent of Shaker 449). Previously we showed that the electric field of the protonated histidines at low extracellular pH (pHe) creates a potential barrier for K+ ions just outside the pore that hinders their exit from the binding site controlling inactivation (control site) thereby slowing inactivation kinetics. Here we examined the effects of extracellular potassium [K+]e and pHe on the rate of inactivation of Kv1.3 using whole-cell patch-clamp. We found that in 150 mM [K+]e inactivation was accelerated upon switching to pHe 5.5 as opposed to the slowing at 5 mM [K+]e. The transition from slowing to acceleration occurred at 40 mM [K+]e, whereas this "turning point" was at 20 mM [K+]e for inward currents. The rate of entry of Ba(2+) ions from the extracellular space to the control site was significantly slowed by low pHe in wild-type hKv1.3, but it was insensitive to pH(e) in H399K and H399L mutants. Based on these observations we expanded our model and propose that the potential barrier created by the protonated histidines impedes the passage of K+ ions between the extracellular medium and the control site in both directions and the effect on inactivation rate (acceleration or slowing) depends on the relative contribution of filling from the extracellular and intracellular sides.     

Protein kinase C modulates inactivation of Kv3.3 channels

Modulation of some Kv3 family potassium channels by protein kinase C (PKC) regulates their amplitude and kinetics and adjusts firing patterns of auditory neurons in response to stimulation. Nevertheless, little is known about the modulation of Kv3.3, a channel that is widely expressed throughout the nervous system and is the dominant Kv3 family member in auditory brainstem. We have cloned the cDNA for the Kv3.3 channel from mouse brain and have expressed it in a mammalian cell line and in Xenopus oocytes to characterize its biophysical properties and modulation by PKC. Kv3.3 currents activate at positive voltages and undergo inactivation with time constants of 150-250 ms. Activators of PKC increased current amplitude and removed inactivation of Kv3.3 currents, and a specific PKC pseudosubstrate inhibitor peptide prevented the effects of the activators. Elimination of the first 78 amino acids of the N terminus of Kv3.3 produced noninactivating currents suggesting that PKC modulates N-type inactivation, potentially by phosphorylation of sites in this region. To identify potential phosphorylation sites, we investigated the response of channels in which serines in this N-terminal domain were subjected to mutagenesis. Our results suggest that serines at positions 3 and 9 are potential PKC phosphorylation sites. Computer simulations of model neurons suggest that phosphorylation of Kv3.3 by PKC may allow neurons to maintain action potential height during stimulation at high frequencies, and may therefore contribute to stimulus-induced changes in the intrinsic excitability of neurons such as those of the auditory brainstem.     

Role of Kv1.3 mitochondrial potassium channel in apoptotic signalling in lymphocytes

Mitochondria have been shown to play a pivotal role in apoptotic signalling in various cell types. We have recently reported that in lymphocytes the voltage-gated potassium channel Kv1.3, known to reside in the plasma membrane, is active also in the inner mitochondrial membrane. Upon induction of apoptosis, outer-membrane inserted Bax binds to and inhibits Kv1.3 resulting in hyperpolarization, an increase in reactive oxygen species production and cytochrome c release. In cells lacking Kv1.3 these events do not take place. Here, we present new data which further corroborates an important role of this channel in the sequence of events leading to Bax-induced cytochrome c release. Recombinant Kv1.3, when pre-incubated with Bax, prevents the actions of Bax at the level of mitochondria. Furthermore, we report the presence of Kv1.3 protein in mitochondria from PC3 and MCF-7 cancer cells, suggesting that this channel might play a role in the apoptotic signalling not only in lymphocytes but also in other cells.     

Phosphorylation is required for alteration of kv1.5 K(+) channel function by the Kvbeta1.3 subunit.

The Kv1.5 K(+) channel is functionally altered by coassembly with the Kvbeta1.3 subunit, which induces fast inactivation and a hyperpolarizing shift in the activation curve. Here we examine kinase regulation of Kv1.5/Kvbeta1.3 interaction after coexpression in human embryonic kidney 293 cells. The protein kinase C inhibitor calphostin C (3 microM) removed the fast inactivation (66 +/- 1.9 versus 11 +/- 0.25%, steady state/peak current) and the beta-induced hyperpolarizing voltage shift in the activation midpoint (V(1/2)) (-21.9 +/- 1.4 versus -4.3 +/- 2.0 mV). Calphostin C had no effect on Kv1.5 alone with respect to inactivation kinetics and V(1/2). Okadaic acid, but not the inactive derivative, blunted both calphostin C effects (V(1/2) = -17.6 +/- 2.2 mV, 38 +/- 1.8% inactivation), consistent with dephosphorylation being required for calphostin C action. Calphostin C also removed the fast inactivation (57 +/- 2.6 versus 16 +/- 0.6%) and the shift in V(1/2) (-22.1 +/- 1.4 versus -2.1 +/- 2.0 mV) conferred onto Kv1.5 by the Kvbeta1.2 subunit, which shares only C terminus sequence identity with Kvbeta1. 3. In contrast, modulation of Kv1.5 by the Kvbeta2.1 subunit was unaffected by calphostin C. These data suggest that Kvbeta1.2 and Kvbeta1.3 subunit modification of Kv1.5 inactivation and voltage sensitivity require phosphorylation by protein kinase C or a related kinase.     

Kv1.3 potassium channel-blocking toxin Ctri9577, novel gating modifier of Kv4.3 potassium channel from the scorpion toxin family

Scorpion toxin Ctri9577, as a potent Kv1.3 channel blocker, is a new member of the α-KTx15 subfamily which are a group of blockers for Kv4.x potassium channels. However, the pharmacological function of Ctri9577 for Kv4.x channels remains unknown. Scorpion toxin Ctri9577 was found to effectively inhibit Kv4.3 channel currents with IC₅₀ value of 1.34 ± 0.03 μM. Different from the mechanism of scorpion toxins as the blocker recognizing channel extracellular pore entryways, Ctri9577 was a novel gating modifier affecting voltage dependence of activation, steady-state inactivation, and the recovery process from the inactivation of Kv4.3 channel. However, Ctri9755, as a potent Kv1.3 channel blocker, was found not to affect voltage dependence of activation of Kv1.3 channel. Interestingly, pharmacological experiments indicated that 1 μM Ctri9755 showed less inhibition on Kv4.1 and Kv4.2 channel currents. Similar to the classical gating modifier of spider toxins, Ctri9577 was shown to interact with the linker between the transmembrane S3 and S4 helical domains through the mutagenesis experiments. To the best of our knowledge, Ctri9577 was the first gating modifier of potassium channels among scorpion toxin family, and the first scorpion toxin as both gating modifier and blocker for different potassium channels. These findings further highlighted the structural and functional diversity of scorpion toxins specific for the potassium channels.     

A high-Na(+) conduction state during recovery from inactivation in the K(+) channel Kv1.5

Na(+) conductance through cloned K(+) channels has previously allowed characterization of inactivation and K(+) binding within the pore, and here we have used Na(+) permeation to study recovery from C-type inactivation in human Kv1.5 channels. Replacing K(+) in the solutions with Na(+) allows complete Kv1.5 inactivation and alters the recovery. The inactivated state is nonconducting for K(+) but has a Na(+) conductance of 13% of the open state. During recovery, inactivated channels progress to a higher Na(+) conductance state (R) in a voltage-dependent manner before deactivating to closed-inactivated states. Channels finally recover from inactivation in the closed configuration. In the R state channels can be reactivated and exhibit supernormal Na(+) currents with a slow biexponential inactivation. Results suggest two pathways for entry to the inactivated state and a pore conformation, perhaps with a higher Na(+) affinity than the open state. The rate of recovery from inactivation is modulated by Na(+)(o) such that 135 mM Na(+)(o) promotes the recovery to normal closed, rather than closed-inactivated states. A kinetic model of recovery that assumes a highly Na(+)-permeable state and deactivation to closed-inactivated and normal closed states at negative voltages can account for the results. Thus these data offer insight into how Kv1. 5 channels recover their resting conformation after inactivation and how ionic conditions can modify recovery rates and pathways.     

Molecular modeling of interactions of agitoxin 2 with Kv1.3 voltage-gated potassium channel

The Kv1.3 voltage-gated potassium channel is involved in a number of processes in excitable and nonexcitable cells: maintenance of resting membrane potential, signal transduction, apoptosis, regulation of cell volume, activation and proliferation of white blood cells. Blocking this channel is an effective approach for the treatment of autoimmune, oncological, chronic inflammatory, and metabolic diseases. The most prospective blockers of Kv1.3 are toxins isolated from the venom of scorpions. Knowledge of the molecular aspects of binding of peptide blockers with the channel is an important condition for the creation of highly effective and selective ligands. In the present work, a complex of hybrid channel KcsA-Kv1.3 with agitoxin 2 was built using homology modeling and molecular dynamics simulation. Analysis of formed contacts allowed us to reveal a complete pattern of interactions and to identify key residues that are responsible for the toxin binding affinity. Results of computational experiment are consistent with the experimental data and important for drug development.